dc.contributor.author
Speck, David
dc.contributor.author
Kleinau, Gunnar
dc.contributor.author
Meininghaus, Mark
dc.contributor.author
Erbe, Antje
dc.contributor.author
Einfeldt, Alexandra
dc.contributor.author
Szczepek, Michal
dc.contributor.author
Scheerer, Patrick
dc.contributor.author
Pütter, Vera
dc.date.accessioned
2022-03-24T07:30:39Z
dc.date.available
2022-03-24T07:30:39Z
dc.identifier.uri
https://refubium.fu-berlin.de/handle/fub188/34491
dc.identifier.uri
http://dx.doi.org/10.17169/refubium-34209
dc.description.abstract
G-protein coupled receptors (GPCR) transduce extracellular stimuli into the cell interior and are thus centrally involved in almost all physiological-neuronal processes. This essential function and association with many diseases or pathological conditions explain why GPCRs are one of the priority targets in medical and pharmacological research, including structure determination. Despite enormous experimental efforts over the last decade, both the expression and purification of these membrane proteins remain elusive. This is attributable to specificities of each GPCR subtype and the finding of necessary experimental in vitro conditions, such as expression in heterologous cell systems or with accessory proteins. One of these specific GPCRs is the leucine-rich repeat domain (LRRD) containing GPCR 7 (LGR7), also termed relaxin family peptide receptor 1 (RXFP1). This receptor is characterized by a large extracellular region of around 400 amino acids constituted by several domains, a rare feature among rhodopsin-like (class A) GPCRs. In the present study, we describe the expression and purification of RXFP1, including the design of various constructs suitable for functional/biophysical studies and structure determination. Based on available sequence information, homology models, and modern biochemical and genetic tools, several receptor variations with different purification tags and fusion proteins were prepared and expressed in Sf9 cells (small-scale), followed by an analytic fluorescence-detection size-exclusion chromatography (F-SEC) to evaluate the constructs. The most promising candidates were expressed and purified on a large-scale, accompanied by ligand binding studies using surface plasmon resonance spectroscopy (SPR) and by determination of signaling capacities. The results may support extended studies on RXFP1 receptor constructs serving as targets for small molecule ligand screening or structural elucidation by protein X-ray crystallography or cryo-electron microscopy.
en
dc.rights.uri
https://creativecommons.org/licenses/by/4.0/
dc.subject
G-protein coupled receptors (GPCR)
en
dc.subject
leucine-rich repeat containing receptor 7 (LGR7)
en
dc.subject
relaxin family peptide receptor 1 (RXFP1)
en
dc.subject
fluorescence-detection size-exclusion chromatography (FSEC)
en
dc.subject
protein engineering
en
dc.subject
surface plasmon resonance spectroscopy (SPR)
en
dc.subject.ddc
600 Technik, Medizin, angewandte Wissenschaften::610 Medizin und Gesundheit::610 Medizin und Gesundheit
dc.title
Expression and Characterization of Relaxin Family Peptide Receptor 1 Variants
dc.type
Wissenschaftlicher Artikel
dcterms.bibliographicCitation.articlenumber
826112
dcterms.bibliographicCitation.doi
10.3389/fphar.2021.826112
dcterms.bibliographicCitation.journaltitle
Frontiers in Pharmacology
dcterms.bibliographicCitation.originalpublishername
Frontiers Media SA
dcterms.bibliographicCitation.volume
12
refubium.affiliation
Charité - Universitätsmedizin Berlin
refubium.resourceType.isindependentpub
no
dcterms.accessRights.openaire
open access
dcterms.bibliographicCitation.pmid
35153771
dcterms.isPartOf.eissn
1663-9812